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So, these 4 proteins are bona fide RNase P proteins, and I'll tell you later that these are the only proteins that are required for catalytic function in M.thermoautotrophicus. How are they put together? We have identified the protein:protein interactions in the archaeal RNase P genetically, using the yeast 2-hybrid system. Most of you are probably familiar with this method, but it is based on yeast strains in which some reporter gere (we used His3 and LacZ) is controlled by the the LexA promoter. Your genes of interest are cloned separately as fusions with the DNA-binding domain of the LexA transcription factor on one hand, and with the transcriptional activator domain of of the GAL4 transcription factor on the other. The first protein is localized to the LexA promoter by the LexA DNA-binding domain. If, and only if, the two proteins being tested interact, the GAL4 activator domain is brought to the promoter, driving transcription of the reporter gene. In the case of the LacZ reporter, interaction of the two test proteins results in the yeast becoming lac+; in the case of the His3 reporter, the cells become His+, they can grow in the absence of histidine (they can make their own) and are resistant to low levels of the his-biosynthetic inhibitor 3-aminotriazol (this helps overcome some leakiness of the his phenotype).